This NRSA Predoctoral Fellowship will provide Lawrence C. Blume with research training under the guidance of Dr. Allyn C. Howlett in the Department of Physiology and Pharmacology at Wake Forest University Health Sciences. Cannabis is currently the most widely used illicit drug in the United Sates, and produces its psychotropic effects primarily through CB1 cannabinoid receptors in the central nervous system. CB1 receptors can be regulated by protein-protein interactions with a variety of molecules, which can lead to alterations in sub-cellular localization, receptor activation and signal transduction. The discovery of CRIP1a, a protein that binds to the distal C-terminus of CB1, has been implicated in the regulation of constitutive (unstimulated by agonists) activity of these receptors. Research findings from my first laboratory rotation indicated that CRIP1a over-expression in rat striatum is influenced by CB1 and D2 receptor levels, and CRIP1a significantly modulates expression of opioid peptides and their receptors (submitted for publication). Because it appears that CRIP1a is an integral regulator of CB1 function in neurons, the remainder of my dissertation will focus on the biochemical and cellular mechanisms of the CRIP1a-CB1 interaction using cultured neuronal cells. Currently, CRIP1a stably over-expressing and knock-down neuronal cell lines have been developed. In neuronal cells, my work demonstrates that CRIP1a over-expression modulates CB1 receptor localization, G protein activation, and constitutive activity in downstream signaling to extra-cellular signal-regulated kinase (ERK). These findings form the basis for the investigation of three specific aims in this application that will elucidate the mechanisms by which CRIP1a modulates CB1 receptor functioning. Aim 1 will use biochemical techniques to establish the role of CRIP1a in CB1 translocation to the plasma membrane and internalization during agonist and antagonist conditions. Aim 2 will determine how CRIP1a affects CB1-G protein-mediated signal transduction using [35S]GTP-gamma-S binding, cAMP accumulation, and ERK phosphorylation in CRIP1a over- expressing and knock-down cells. Aim 3 will assess the potential for the CB1-CRIP1a complex to interact with PDZ domain-containing scaffolding proteins using pull-down and co-immunoprecipitation experiments. These studies will provide valuable information regarding CRIP1a's ability to modulate CB1 functioning and potentially lead to the development of novel therapeutic strategies based on selectively fine-tuning CB1 receptor function. Because CRIP1a reverses the constitutive activity of CB1 receptors in neuronal cells, it is plausible to develop drugs that block this interaction, and in turn enhance CB1 activity. The diversity of experimental approaches proposed to address these focused research efforts will provide an outstanding pre-doctoral training experience to the applicant in support of his career goals as an independent scientist. PUBLIC HEALTH RELEVANCE: CB1 cannabinoid receptors are abundant in the brain, and are therapeutic targets for many diverse medical conditions, including metabolic syndrome, pain, glaucoma, nausea, cancer and substance abuse disorders. CRIP1a is a novel accessory protein that binds to CB1 receptors, and modulates CB1 receptor expression, trafficking and signaling. This research investigation will facilitate the development of small-drug molecules capable of specifically altering the CRIP1a-CB1 interaction, and providing a new means of treatment for disorders for which CB1 receptor regulation has therapeutic potential (e.g., metabolic syndrome, chronic pain, epilepsy, drug addiction).